JP2011512408A - Composition for increasing viral vector uptake into the myocardium - Google Patents

Composition for increasing viral vector uptake into the myocardium Download PDF

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JP2011512408A
JP2011512408A JP2010547611A JP2010547611A JP2011512408A JP 2011512408 A JP2011512408 A JP 2011512408A JP 2010547611 A JP2010547611 A JP 2010547611A JP 2010547611 A JP2010547611 A JP 2010547611A JP 2011512408 A JP2011512408 A JP 2011512408A
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therapeutic polynucleotide
infusion
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nitroglycerin
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クリスティーナ・マリア・ゼボ
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セラドン・コーポレーション
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/04Nitro compounds
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
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    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N2750/14011Parvoviridae
    • C12N2750/14111Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141Use of virus, viral particle or viral elements as a vector
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    • C12N2750/14011Parvoviridae
    • C12N2750/14111Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14171Demonstrated in vivo effect
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y10S977/00Nanotechnology
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    • Y10S977/904Specified use of nanostructure for medical, immunological, body treatment, or diagnosis
    • Y10S977/908Mechanical repair performed/surgical
    • Y10S977/913Stem cell therapy implantation

Abstract

  The present invention relates to improved therapies for treating heart disease, particularly improved delivery of therapeutic agents to heart tissue by direct injection into the coronary circulation. A preferred embodiment of the present invention is a method of treating or preventing cardiovascular disease by transfecting the heart cells of a large mammal, identifying a mammal in need of treatment or prevention of heart disease, Injecting therapeutic polynucleotide into the blood vessel for at least about 3 minutes prior to and / or during the infusion of the therapeutic polynucleotide into the coronary circulation in vivo. However, the coronary circulation is not separated or substantially separated from the mammalian systemic circulation, and the heart disease is treated or prevented by transfecting the animal's heart cells with a therapeutic polynucleotide.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is claimed in priority under 35 USC 119 (e) (1), the application of which is incorporated herein by reference in its entirety, 2008 This is a non-provisional application of US Provisional Patent Application No. 61 / 029,881 filed on February 19th.

  The present invention relates to gene therapy for treating heart disease, in particular to increased delivery of polynucleotides to heart tissue.

  Heart disease is a major public health problem with a very high prevalence, especially in Western countries. Heart diseases include coronary artery disease, ischemic heart disease, angina pectoris, heart failure, valvular heart disease, cardiac arrhythmia and heart inflammation (myocarditis), to name a few. Coronary artery disease and heart failure are probably the most serious and prevalent, and are the leading causes of death in Western countries. The impact of acute myocardial infarction and congestive heart failure and their sequelae on patient quality of life and health care costs will search for new therapies.

  Heart failure (HF) is a serious disease that causes the heart to lose its ability to pump blood efficiently. Data from the National Heart, Lung and Blood Institute suggest that about 5 million people in the United States alone have heart failure and an additional 550,000 new cases are diagnosed each year. HF causes or causes about 300,000 deaths per year. The disease is most common in people over the age of 65, women and African Americans. The most common symptoms of heart failure are shortness of breath, fatigue, and swelling of the ankles, feet, legs, and sometimes the abdomen. There is no cure for congestive heart failure, and there is a clear need in the art for effective therapy.

  One method of treating heart disease, such as HF, that is starting to gain more attention is gene therapy, which delivers polynucleotides in heart tissue, typically viral vectors. Direct injection into the myocardium (Liu et al., FASEB J. 2006; 20 (2): 207-16; Liu et al. Toxicol. Appl. Pharmacol. 2006 Jan 25 (electronic publication); Zhu et al., Circulation 2005; 112 (17): 2650-9), intracoronary delivery (Nykanen et al., Circ. Res. 2006; 98 (11): 1373-80; Kaspar et al., J. Gene Med. 2005; 7 (3): 316-24), catheter-based antegrade intracoronary delivery while blocking the coronary vein (Hayase et al., Am. J. Physiol. Heart Circ. Physiol. 2005; 288 (6): H2995-3000), followed by aortic and pulmonary artery blockade, followed by proximal aortic injection of adeno-associated virus vector (Kaspar et al., J. Gene Med. 2005; 7 (3): 316-24) A number of means of delivering viral vectors have been tried. Leiden and Svensson generally refer to in vivo injection of rAAV vectors into coronary arteries or sinuses, but perfusion of mouse hearts with reporter genes ex vivo at 4 ° C when the heart stopped beating (WO00 / 38518), only those methods that are impractical for treating large mammals such as humans are described in detail.

  Therefore, these methods are too dangerous, for example, because of the need for surgical intervention or because the flow of oxygen-containing blood into the myocardium is blocked, so that the viral vectors needed to carry out the method Due to the volume, the low percentage of tissue transfected, due to the fact that transduction is limited only to the injection / dose site, or the method is impractical or unconfirmed for the treatment of disease in large animals or humans Therefore, neither is sufficient for use in a clinical setting. There remains a need for a concise, minimally invasive but effective means of using viral vectors to deliver transgenes to heart tissue to treat disease, particularly in humans.

  For example, in US Patent Application No. 11 / 778,900, previously incorporated herein by reference in its entirety, a method of transfection of cardiac cells using slow infusion of therapeutic polynucleotides into coronary vessels Is described. Increasing the effectiveness of transfection of polynucleotides into heart cells may also increase the effectiveness of treatment.

  The use of nitroglycerin as part of a pre-treatment cocktail has been used in animal myocardial gene transfer therapy (described in porcine experiments with myocardial gene transfer using viral vectors, the entire contents of which are incorporated by reference. (See Sasano T., et al., "Targeted High-Efficiency, Homogeneous Myocardial Gene Transfer," J. Mol. Cell. Cardiol., 2007 May; 42 (5): 954-961, incorporated herein) . As part of efforts to increase the effectiveness of gene transfer in healthy pigs, they use pre-therapeutic cocktails containing vascular endothelial growth factor (VEGF), nitroglycerin, adenosine and calcium, then viral vectors and the aforementioned Report administration of active agent combinations. However, such protocols are not clinically practical because their treatment protocols can lead to extreme hypotension and cardiac side effects. Sasano, for example, “Pre-treatment and infusion of virus solution caused blood pressure drop due to rapid cardiac contraction of 30 mmHg and stabilized within 1 minute of perfusion. Average heart rate also decreased by 50-60 / min, then the same It has stabilized over time. " The authors stated that `` 5 (50%) of the first 10 pigs and 4 (5.6%) of the remaining 71 pigs had ventricular fibrillation (VF) during intracoronary infusion. ) Occurred ", further reported (Sasano et al, page 958). Given the frailty state of most human subjects with advanced heart disease, these side effects are likely to be even less tolerated than young healthy animals.

US Provisional Patent Application No. 61 / 029,881 WO00 / 38518 U.S. Patent Application No. 11 / 778,900 U.S. Pat.No. 5,139,941 U.S. Pat.No. 4,797,368

Liu et al., FASEB J. 2006; 20 (2): 207-16 Liu et al. Toxicol. Appl. Pharmacol. 2006 Jan 25 (electronic publication) Zhu et al., Circulation. 2005; 112 (17): 2650-9 Nykanen et al., Circ. Res. 2006; 98 (11): 1373-80 Kaspar et al., J. Gene Med. 2005; 7 (3): 316-24 Hayase et al., Am. J. Physiol. Heart Circ. Physiol. 2005; 288 (6): H2995-3000 Sasano T., et al., "Targeted High-Efficiency, Homogeneous Myocardial Gene Transfer" J. Mol. Cell. Cardiol., 2007 May; 42 (5): 954-961 Li et al., Molecular Therapy vol. 16 no. 7 July 2008, pg. 1252-1260 Jeremy M. Berg, et al. (2006), Biochemistry, 6th Edition. W. H. Freeman and Company Megson IL, Webb DJ, "Nitric oxide donor drugs: current status and future trends" Expert. Opin. Investig. Drugs, 2002 May; 11 (5): 587-601 Vlodaver Z. et al. Coronary Heart Disease: Clinical, Angiographic, and Pathologic Pofiles. Spinger-Verlag, New York. 1976 McAlpine W. Heart and Coronary Ateries.Spinger Verlag, 1975 Flotte et el., Proc. Natl. Acad. Sci. USA, 1993; 90: 10613-17 Walsh et al., J Clin. Invest., 1994; 94: 1440-48 Crameri, A et al. (1998) .``DNA shuffling of a family of genes from diverse species accelerates directed evolution. '' Nature 391: 288-291 Stemmer, WP (1994) .Rapid evolution of a protein in vitro by DNA shuffling.Nature 370: 389-391 Moore, G. L., Maranas, C. D., 2000. "Modeling DNA mutation and recombination for directed evolution experiments." J. Theor. Biol. 205, 483-503 Maheshri, N et al. (2006). "Directed evolution of adenoassociated virus yields enhanced gene delivery vectors." Nat Biotechnol 24: 198-204 Hajjar et al., Journal of Cardiac Failure, 2008 14 (5); 355-67

  Thus, increasing the effectiveness of transfection of heart cells using viral vectors such as vasodilators and adeno-associated virus (AAV) that can be used in clinical settings that do not result in life-threatening hypotension or cardiac arrhythmias There is still a need to develop treatment methods for this.

  The present invention relates to uses and therapies for treating heart disease, specifically direct injection or infusion into the coronary arteries, intravenous or subcutaneous, or oral, without the need to impede blood flow. Administration relates to improved or enhanced delivery of therapeutic agents to heart tissue by using vasodilators, preferably nitric oxide (NO) augmenting substances.

  A preferred embodiment of the present invention is a method of treating or preventing cardiovascular disease by transfecting the heart cells of a large mammal, the step of identifying a mammal in need of treatment or prevention of cardiovascular disease Administering to said mammal sufficient vasodilator to dilate coronary blood vessels, and administering the therapeutic polynucleotide to coronary blood vessels in vivo, said therapeutic polynucleotide By injecting into the blood vessel for at least about 3 minutes, wherein the coronary circulation is not separated or substantially separated from the mammalian systemic circulation, and the therapeutic polynucleotide is transfected into the mammalian heart cells. , Wherein the cardiovascular disease is treated or prevented. In some embodiments, the vasodilator is a NO enhancer. In some embodiments, the NO enhancer is nitroglycerin.

  In some embodiments, the NO enhancer is administered to a coronary blood vessel. In some embodiments, the NO enhancing agent is a group consisting of before the injection of the therapeutic polynucleotide, simultaneously with the injection of the therapeutic polynucleotide, and before and simultaneously with the injection of the therapeutic polynucleotide. It is administered in a form selected from In some embodiments, the NO enhancer is administered as a bolus injection within 5 minutes prior to the infusion of the therapeutic polynucleotide.

  In some embodiments, the NO enhancer is administered as a bolus injection within 5 minutes prior to the infusion of the therapeutic polynucleotide, and the NO enhancer is administered in the therapeutic polynucleotide for at least about 10 minutes. The blood vessel is injected simultaneously with the injection.

  In some embodiments, the NO enhancer is about 50 μg to about 150 μg of nitroglycerin.

In some embodiments, administration of the NO enhancer is antegrade of a 1.5 mL 100 μg / mL nitroglycerin solution to at least one of the left or right coronary artery via a percutaneous catheter for less than 1 minute. Including epicardial coronary injection, wherein the administration of the NO enhancer is less than 3 minutes prior to the infusion of the therapeutic polynucleotide, and other vasodilators or vascular permeation enhancers are fed into the mammal Do not administer to animals. In some embodiments, the method further comprises injecting nitroglycerin into the blood vessel simultaneously with the infusion of the therapeutic polynucleotide. In some embodiments, the mammal is a human, the cardiovascular disease is heart failure, and the therapeutic polynucleotide is packaged in DNase resistant particles (DRP) of an AAV2 / 1 viral vector and injected into the blood vessel. The total number of DRPs does not exceed about 1 × 10 13 , the therapeutic polynucleotide comprises a SERCA2a coding sequence, the blood vessel is at least one of the left or right coronary artery, and the infusion of the therapeutic polynucleotide is at least It lasts about 10 minutes. In some embodiments, the treatment is a measure of absolute ejection fraction of the human heart 6 months after the treatment as compared to a measure of absolute ejection fraction of the human heart prior to the treatment. To improve.

  In some embodiments, the NO enhancer is administered systemically. In some embodiments, the NO enhancer is administered systemically in a form selected from the group consisting of intravenous injection, intravenous infusion, oral administration, transdermal administration, and subcutaneous administration.

  In some embodiments, the NO enhancing agent is a group consisting of before the injection of the therapeutic polynucleotide, simultaneously with the injection of the therapeutic polynucleotide, and before and simultaneously with the injection of the therapeutic polynucleotide. It is administered in a form selected from

In some embodiments, about 0.5 mg to about 2.5 mg of nitroglycerin is administered by intravenous infusion for at least 30 minutes prior to the infusion of the therapeutic polynucleotide, the infusion of the therapeutic polynucleotide comprising Beginning within 3 minutes of completing the intravenous infusion of the nitroglycerin, no other vasodilator or vascular penetration enhancer is administered to the mammal. In some embodiments, the method further comprises injecting an additional amount of nitroglycerin simultaneously with the infusion of the therapeutic polynucleotide. In some embodiments, the mammal is a human, the cardiovascular disease is heart failure, and the therapeutic polynucleotide is packaged in DNase resistant particles (DRP) of an AAV2 / 1 viral vector and injected into the blood vessel. The total number of DRPs does not exceed about 1 × 10 13 , the therapeutic polynucleotide comprises a SERCA2a coding sequence, the blood vessel is at least one of the left or right coronary artery, and the infusion of the therapeutic polynucleotide is at least It lasts about 10 minutes. In some embodiments, the treatment is a measure of absolute ejection fraction of the human heart 6 months after the treatment as compared to a measure of absolute ejection fraction of the human heart prior to the treatment. To improve.

  One embodiment of the invention is a therapeutic polynucleotide for use in a method of treating or preventing cardiovascular disease by transfecting a large mammalian heart cell, said method comprising said therapeutic polynucleotide. A therapeutic polynucleotide comprising the step of dilating a blood vessel in the coronary circulation by administering a vasodilatory substance to the mammal prior to and / or simultaneously with the administration. In some embodiments, the method comprises administering a therapeutic polynucleotide to a coronary circulation vessel in vivo, the therapeutic polynucleotide being infused into the vessel for at least about 3 minutes, wherein the coronary circulation is The cardiovascular disease is treated or prevented by transfecting the therapeutic polynucleotide into the mammalian heart cells that are not separated or substantially separated from the mammalian systemic circulation.

  In some embodiments, the vasodilator is a NO enhancer. In some embodiments, the NO enhancing agent is a group consisting of before the injection of the therapeutic polynucleotide, simultaneously with the injection of the therapeutic polynucleotide, and before and simultaneously with the injection of the therapeutic polynucleotide. It is administered in a form selected from

  In some embodiments, the NO enhancer is administered to a coronary blood vessel. In some embodiments, the NO enhancing agent is administered as a bolus injection within 5 minutes prior to the infusion of the therapeutic polynucleotide, and the NO enhancing agent is infused for at least about 10 minutes. At the same time, it is injected into the blood vessel.

  In some embodiments, the NO enhancer is about 50 μg to about 150 μg of nitroglycerin.

In some embodiments, the administration of the NO enhancer is antegrade of a 1.5 mL 100 μg / mL nitroglycerin solution to at least one of the left or right coronary artery via a percutaneous catheter for less than 1 minute Including epicardial coronary injection, the administration of the NO-enhancing substance is less than 3 minutes prior to the infusion of the therapeutic polynucleotide and no other vasodilator or vascular penetration enhancer is administered to the mammal. In some embodiments, the method further comprises injecting nitroglycerin into the blood vessel simultaneously with the infusion of the therapeutic polynucleotide. In some embodiments, the mammal is a human, the cardiovascular disease is heart failure, and the therapeutic polynucleotide is packaged in DNase resistant particles (DRP) of an AAV2 / 1 viral vector and injected into the blood vessel. The total number of DRPs does not exceed about 1 × 10 13 , the therapeutic polynucleotide comprises a SERCA2a coding sequence, the blood vessel is at least one of the left or right coronary artery, and the infusion of the therapeutic polynucleotide is at least It lasts about 10 minutes. In some embodiments, the method of treatment or prevention comprises absolute ejection of the human heart 6 months after the treatment as compared to a measure of the absolute ejection fraction of the human heart prior to the treatment. Improve rate measurements.

In some embodiments, the NO enhancer is administered systemically in a form selected from the group consisting of intravenous injection, intravenous infusion, oral administration, transdermal administration, and subcutaneous administration. In some embodiments, the administration of the NO enhancer comprises administering about 0.5 mg to about 2.5 mg nitroglycerin by intravenous infusion over at least 30 minutes prior to the infusion of the therapeutic polynucleotide. The infusion of the therapeutic polynucleotide begins within 3 minutes of completion of the intravenous infusion of the nitroglycerin and no other vasodilator or vascular penetration enhancer is administered to the mammal. In some embodiments, the method further comprises injecting an additional amount of nitroglycerin simultaneously with the infusion of the therapeutic polynucleotide. In some embodiments, the mammal is a human, the cardiovascular disease is heart failure, and the therapeutic polynucleotide is packaged in DNase resistant particles (DRP) of an AAV2 / 1 viral vector and injected into the blood vessel. The total number of DRPs does not exceed about 1 × 10 13 , the therapeutic polynucleotide comprises a SERCA2a coding sequence, the blood vessel is at least one of the left or right coronary artery, and the infusion of the therapeutic polynucleotide is at least It lasts about 10 minutes. In some embodiments, the method of treatment or prevention comprises absolute ejection of the human heart 6 months after the treatment as compared to a measure of the absolute ejection fraction of the human heart prior to the treatment. Improve rate measurements.

  Another embodiment of the present invention is the use of a therapeutic polynucleotide for the manufacture of a medicament for the treatment or prevention of cardiovascular disease in a large mammal, wherein the therapeutic polynucleotide is a cardiac cell of said large mammal Resulting in the treatment or prevention of the cardiovascular disease, wherein the medicament expands blood vessels in the coronary circulation of the mammal prior to and / or concurrently with the administration of the medicament. Use for combined administration with substances. In some embodiments, the administration of the medicament comprises administering a therapeutic polynucleotide to a coronary circulation vessel in vivo, wherein the therapeutic polynucleotide is infused into the vessel for at least about 3 minutes, Is not separated or substantially separated from the mammalian systemic circulation. In some embodiments, the vasodilator is a nitric oxide (NO) enhancer. In some embodiments, the NO enhancing agent is a group consisting of before the injection of the therapeutic polynucleotide, simultaneously with the injection of the therapeutic polynucleotide, and before and simultaneously with the injection of the therapeutic polynucleotide. It is administered in a form selected from

  In some embodiments, the NO enhancer is administered to a coronary blood vessel. In some embodiments, the NO enhancing agent is administered as a bolus injection within 5 minutes prior to the infusion of the therapeutic polynucleotide, and the NO enhancing agent is infused for at least about 10 minutes. At the same time, it is injected into the blood vessel. In some embodiments, the NO enhancer is about 50 μg to about 150 μg of nitroglycerin.

In some embodiments, the administration of the NO enhancer is antegrade of a 1.5 mL 100 μg / mL nitroglycerin solution to at least one of the left or right coronary artery via a percutaneous catheter for less than 1 minute. Including epicardial coronary injection, the administration of the NO-enhancing substance is less than 3 minutes prior to the infusion of the therapeutic polynucleotide and no other vasodilator or vascular penetration enhancer is administered to the mammal. In some embodiments, the method further comprises injecting nitroglycerin into the blood vessel simultaneously with the infusion of the therapeutic polynucleotide. In some embodiments, the mammal is a human, the cardiovascular disease is heart failure, and the therapeutic polynucleotide is packaged in DNase resistant particles (DRP) of an AAV2 / 1 viral vector and injected into the blood vessel. The total number of DRPs does not exceed about 1 × 10 13 , the therapeutic polynucleotide comprises a SERCA2a coding sequence, the blood vessel is at least one of the left or right coronary artery, and the infusion of the therapeutic polynucleotide is at least It lasts about 10 minutes. In some embodiments, the method of treatment or prevention comprises absolute ejection of the human heart 6 months after the treatment as compared to a measure of the absolute ejection fraction of the human heart prior to the treatment. Improve rate measurements.

In some embodiments, the NO enhancer is administered systemically in a form selected from the group consisting of intravenous injection, intravenous infusion, oral administration, transdermal administration, and subcutaneous administration. In some embodiments, administration of the NO-enhancing agent comprises administering about 0.5 mg to about 2.5 mg nitroglycerin by intravenous infusion over at least 30 minutes prior to the infusion of the therapeutic polynucleotide; The infusion of the therapeutic polynucleotide begins within 3 minutes of completion of the intravenous infusion of nitroglycerin and no other vasodilator or vascular penetration enhancer is administered to the mammal. Some embodiments further comprise injecting an additional amount of nitroglycerin simultaneously with the infusion of the therapeutic polynucleotide. In some embodiments, the mammal is a human, the cardiovascular disease is heart failure, and the therapeutic polynucleotide is packaged in DNase resistant particles (DRP) of an AAV2 / 1 viral vector and injected into the blood vessel. The total number of DRPs does not exceed about 1 × 10 13 , the therapeutic polynucleotide comprises a SERCA2a coding sequence, the blood vessel is at least one of the left or right coronary artery, and the infusion of the therapeutic polynucleotide is at least It lasts about 10 minutes. In some embodiments, the method of treatment or prevention comprises absolute ejection of the human heart 6 months after the treatment as compared to a measure of the absolute ejection fraction of the human heart prior to the treatment. Improve rate measurements.

  In some embodiments, the NO enhancer comprises nitroglycerin. In some embodiments, the NO enhancer consists essentially of nitroglycerin. In some embodiments, the NO enhancer comprises nitroglycerin. In some embodiments, no other vasodilator or vascular penetration enhancer is administered to the mammal.

  In some embodiments, coronary circulation outflow is not unnaturally restricted.

  In some embodiments, transfection of cardiac cells in the anterior lateral ventricle, lower lateral ventricle, septum and right ventricle can be detected using quantitative PCR (RNA or DNA).

  In some embodiments, the polynucleotide can express a protein capable of modulating cellular activity of heart cells. In some embodiments, the cellular activity is the calcium circulation pathway of cardiomyocytes. In some embodiments, the protein is sarcoplasmic reticulum / endoplasmic reticulum ATPase (SERCA). In some embodiments, the SERCA is SERCA2a.

  In some embodiments, the polynucleotide is a viral vector selected from the group consisting of adeno-associated virus, adenovirus, retrovirus, herpes simplex virus, bovine papilloma virus, lentiviral vector, vaccinia virus, and polyoma virus. Present in. In some embodiments, the viral vector is an AAV virus. In some embodiments, the viral vector is an AAV virus that includes a heterologous capsid protein such that not all of the capsid proteins VP1, VP2, and VP3 are AAV of the same serotype. In some embodiments, the heterologous capsid protein comprises capsid proteins from AAV1 and AAV2. In some embodiments, the viral vector is an AAV2 / 1 vector. In some embodiments, the polynucleotide is operably linked to a CMV-based promoter and packaged in the viral vector. In some embodiments, the polynucleotide comprises a SERCA2a coding sequence.

  In some embodiments, the transfection of the heart cells increases lateral ventricular shortening rate. In some embodiments, the mammal is a human and the disease is congestive heart failure. In some embodiments, the transfection of the cardiac cells is at least 25% lateral ventricular shortening as measured about 4 months after the injection, as compared to the lateral ventricular shortening prior to polynucleotide injection. Increase. In some embodiments, the transfection of the cardiac cells is a heart selected from the group consisting of SERCA2a protein expression, shortening rate, ejection fraction, cardiac output, ventricular relaxation time constant, and reverse flow Bring improvements in functional measurements.

  In some embodiments, the infusion into the blood vessel is at a rate of about 6.0 mL / min or less, in some embodiments it is at a rate of about 2.5 mL / min or less, in some embodiments, It is at a rate of about 2.0 mL / min or less, in some embodiments it is a rate of about 1.2 mL / min or less, in some embodiments it is a rate of about 1.0 mL / min or less, In some embodiments, it is at a rate of about 0.6 mL / min or less.

  In a preferred embodiment, the polynucleotide is present in a viral vector selected from the group consisting of adeno-associated virus, adenovirus, retrovirus, herpes simplex virus, bovine papilloma virus, lentiviral vector, vaccinia virus, and polyoma virus. To do. In a more preferred embodiment, the viral vector is an AAV virus or AAV molecular variant (Li et al., Molecular Therapy vol. 16 no. 7 July 2008, the entire contents of which are incorporated herein by reference. pg. 1252-1260), in a more preferred embodiment, the viral vector is an AAV2 / 1 vector. The AAV2 / 1 vector consists of inverted terminal repeats (ITR) from AAV serotype 1 capsid and AAV serotype 2. In some embodiments, the polynucleotide is operably linked to a CMV-based promoter and packaged in a viral vector. In a preferred embodiment, the polynucleotide comprises human SERCA2a cDNA. Preferably, the vector consists of AAV serotype 1 capsid and contains human SERCA2a cDNA flanked by inverted terminal repeats (ITR) from AAV serotype 2 (AAV1 / SERCA2a).

A preferred embodiment of the present invention is a method of treating or preventing heart disease by transfecting large mammalian heart cells, wherein 50-150 micrograms of intracoronary bolus injection lasting less than 1 minute into coronary arterial vessels. Injecting nitroglycerin in vivo, injecting about 1.4 × 10 11 to about 1 × 10 13 DRP of AAV1 / SERCA2a into coronary circulation vessels in vivo, with AAV1 / SERCA2a in the vessel for at least about 3 minutes A method in which heart disease is treated or prevented by infusion and transfusion of AAV1 / SERCA2a into mammalian heart cells, where the coronary circulation is not separated or substantially separated from the mammalian systemic circulation . In a preferred embodiment, nitroglycerin is the only vasodilator or permeation enhancer that is administered and no other vasodilator or permeation enhancer is administered.

FIG. 6 is a cross-sectional view of the heart tested for SERCA2a expression in normal Gottingen piglets. (A) Left ventricle (LV) basal layer, (B) LV intermediate layer, (C) LV apical layer. FIG. 30 is a diagram of results from a single dose, cardiac distribution study on day 30 of direct intra-coronary infusion of AAV1 / SERCA2a in normal Gottingen piglets. The graph demonstrates the expression of SERCA2a mRNA by RT-PCR in the heart cross section at 1 month follow-up from groups 1 and 3. FIG. 14 is a diagram of results from a single dose, tissue distribution study on day 30 of direct intra-coronary infusion of AAV1 / SERCA2a in normal Gottingen piglets. The graph demonstrates the expression of SERCA2a protein in the heart cross section at 1 month follow-up from groups 1, 2, 3 and 4. FIG. 6 is a graph of mean aortic pressure from groups 3 and 4 during the experimental procedure for administration of AAV1 / SERCA2a in normal Gottingen piglets.

  The technology of the present invention relates to improved use and therapy for treating heart disease, specifically the delivery of therapeutic and vasodilators to heart tissue that does not require blocking blood flow. A preferred embodiment of the present invention is a method of treating or preventing heart disease by transfecting the heart cells of a large mammal, the step of identifying a mammal in need of treatment or prevention of heart disease, preferably Includes injecting or injecting a substance to increase vasodilation by increasing the amount of nitric oxide in the coronary circulation, injecting therapeutic polynucleotides into coronary blood vessels in vivo, By infusing the therapeutic polynucleotide into the blood vessel for at least about 3 minutes, wherein the coronary circulation is not separated or substantially separated from the mammalian systemic circulation, and the therapeutic polynucleotide is transfected into mammalian cardiac cells A method by which heart disease is treated or prevented.

  As used herein, “polynucleotide” has its normal and customary meaning in the art and includes any polymeric nucleic acid, such as a DNA or RNA molecule, and chemical derivatives known to those skilled in the art. Polynucleotides not only include polynucleotides that encode therapeutic proteins, but also include expression of target nucleic acid sequences (e.g., antisense, interference, or small interfering nucleic acids) using techniques known in the art. Also included are sequences that can be used to reduce. An example is a sequence that reduces or eliminates the expression of phospholamban. Polynucleotides can also be used to initiate or augment target nucleic acid sequence expression or target protein production in the cardiovascular system. Target nucleic acids and proteins include nucleic acids and proteins normally found in the target tissue (including naturally occurring mutations), derivatives of such naturally occurring nucleic acids or proteins, and naturally present that are not normally found in the target tissue There are, but are not limited to, nucleic acids or proteins, or synthetic nucleic acids or proteins. One or more polynucleotides can be combined and administered simultaneously and / or sequentially to increase and / or decrease one or more target nucleic acid sequences or proteins.

  As used herein, the terms “infusion”, “injected”, and “inject” have their usual and customary meaning in the art, and the term “injection” recognized in the art. Or refers to administration over a period of time (typically 1 minute or more) substantially longer than a “bolus injection” (typically less than 1 minute). The infusion flow rate should depend at least in part on the volume to be administered, however, the “infusion” flow rate is slower than the “injection” flow rate of the same volume.

  “Effective amount” has its ordinary and customary meaning in the art and includes an amount sufficient to effect or achieve a beneficial or desired therapeutic effect. For example, an `` effective amount '' achieves any of the following: increased lateral ventricular shortening rate and / or pain relief, improvement, stabilization, reversal, progression of disease state or slowing or delaying signs or symptoms Amount. An effective amount can be administered in one or more administrations.

  As used herein, “with”, “in combination with”, “simultaneous” or “simultaneously” has their usual and customary meaning in the art, and implementation of one treatment modality and other treatment modalities including. For example, in addition to administration of one or more pharmaceutical compositions to the same individual, injection of the polynucleotide into the subject can be performed. As used herein, these terms include simultaneous or near simultaneous administration.

  The disclosed methods and therapeutic agents disclosed herein can be combined with existing therapies for heart disease, including those previously listed in the introductory portion, such as drugs and transcutaneous or surgical interventions. Compared with the treatment alone, it can provide a high therapeutic effect. A high therapeutic effect is, for example, an extension of the time period between worsening signs or symptoms of disease compared to the average or typical time period for an existing treatment regimen, or average or typical for standard treatment alone This can be demonstrated by an extension of the time required before additional treatment is required compared to the time required.

As used herein, “treating” a disease or “treating” a disease has its usual and customary meaning in the art, including the progression of the disease or the cessation or slowing of signs or symptoms of the disease Including stable state of disease, healing, incomplete healing. The term “prevention” has its usual and customary meaning in the art and includes complete or incomplete prevention of disease or signs or symptoms of the disease, delayed onset. The term “therapeutic”, “therapeutic effect” or “clinical effect” includes both treatment and prevention. Examples of diseases associated with the cardiovascular system that may be treated using the techniques of the present invention include heart failure, ischemia, arrhythmia, myocardial infarction, congestive heart failure, graft rejection, abnormal cardiac contraction Sexual, non-ischemic cardiomyopathy, mitral regurgitation, aortic stenosis or reflux, abnormal Ca 2+ metabolism and congenital heart disease, but are not limited to these. For example, beneficial or desired clinical outcome or therapeutic effect may include increased survival, significant reduction in signs or symptoms of cardiovascular disease, further reduction in the extent of the disease, stable (ie, does not worsen) disease state, disease There is, but is not limited to, delay or slowing of progression, improvement of disease state or relief of pain, and remission (whether partial or total), detectable or undetectable. Other examples of therapeutic effects include increased lateral ventricular shortening rate, increased myocardial contractility at the cellular and intact animal level, reversal of cardiac remodeling, and normalization of abnormally high cardiac dilation levels of cytoplasmic calcium. There are, but not limited to. Other clinical features that can be improved in subjects treated with embodiments of the invention include, but are not limited to, survival, cardiac metabolism, myocardial contractility, heart rate, ventricular function (e.g., left ventricular ejection fraction). Fraction (LVEF), left ventricular end systolic volume (LVESV), left ventricular end diastolic pressure (LVEDP), left ventricular end systolic pressure (LVSP)), Ca 2+ metabolism (e.g., intracellular Ca 2+ concentration, peak or Resting state [Ca 2+ ], SR Ca 2+ ATPase activity, phosphorylation state of phospholamban), force generation, cardiac relaxation and pressure, force contraction frequency relationship, cardiac cell survival or apoptosis or ion channel activity (eg Sodium and calcium exchange, sodium channel activity, calcium channel activity, sodium potassium ATPase pump activity), myosin heavy chain, troponin I, troponin C, troponin T, tropomyosin, actin, myosin light chain kinner Z, myosin light chain 1, myosin light chain 2 or myosin light chain 3, IGF-1 receptor, PI3 kinase, AKT kinase, sodium-calcium exchanger, calcium channels (L and T), calsequestrin, calreticulin Inhibitor-1 of type 1 protein phosphatase, or any agent that promotes dephosphorylation of phospholamban, or an inhibitor of sarcoplasmic reticulum calcium pump (SERCA2a). Other measures of heart disease that can be improved include shortening rate, cardiac output, ejection fraction, Tau, backflow, short-term hospitalization, improved quality of life, increased treadmill time, 6 Includes increased distance during the minute walk test and increased maximum oxygen consumption (VO 2 max).

  As used herein, an “exogenous” nucleic acid or gene is a nucleic acid or gene that is not naturally present in the vector utilized for nucleic acid transfer, eg, not found in a viral vector, although the term It is not intended to exclude nucleic acids encoding proteins or polypeptides naturally present therein, such as SERCA.

  As used herein, a “cardiac cell” is any heart that is involved in maintaining the structure of the heart or providing cardiac function, such as cardiomyocytes, cells of the cardiovascular system, or cells present in the heart valve Including cells. Cardiac cells include cardiomyocytes (having both normal and abnormal electrical properties), epithelial cells, endothelial cells, fibroblasts, tract tissue cells, cardiac pacemaker cells, and neurons.

  As used herein, “isolated”, “substantially isolated” or “mostly isolated” and variations thereof are fully or absolute of coronary veins, heart, systemic veins, or systemic circulation Are terms that do not require a general separation, but rather they shall mean that most, preferably the main or even substantially all of the specific circulation is separated. As used herein, “partially separated” refers to any significant portion of the particular cycle being separated.

  As used herein, `` unnaturally restricted '' includes any method that restricts fluid flow through a blood vessel, such as balloon catheters, sutures, etc., but naturally occurring restrictions such as plaque accumulation ( (Stenosis) is not included. Unnatural limitations include, for example, substantial or complete separation of the coronary circulation.

  As used herein, “modulate” has its ordinary meaning and includes both increasing and decreasing target expression or activity.

  As used herein, the term “minimally invasive” is intended to include any procedure that does not require a surgical incision procedure into the heart or a blood vessel closely associated with the heart. Such procedures also include the use of endoscopic means to access the heart and catheter system means that rely on access through the aorta and veins, femoral artery, and the like.

  As used herein, the term “adeno-associated virus” or “AAV” includes all subtypes, serotypes and pseudotypes, as well as natural and recombinant or molecular variants (see Li et al). A variety of AAV serotypes and strains are known in the art and are generally available from sources such as ATCC and professional or commercial sources. Alternatively, sequences derived from serotypes and strains of AAV that are published and / or available from various databases can be synthesized using known techniques.

  The term “serotype” as used herein refers to an AAV that is identified and distinguished from other AAVs based on the reactivity of the capsid protein with a distinct antiserum. There are at least 12 known human AAV serotypes, including AAV1-AAV12, however other serotypes continue to be discovered and the use of newly discovered serotypes is contemplated. For example, an AAV2 serotype is used to refer to an AAV that includes the encoded capsid protein from the AAV2 cap gene and a genome that includes 5 ′ and 3 ′ inverted terminal repeat (ITR) sequences from the same AAV2 serotype.

  “Pseudotype” AAV refers to an AAV comprising a capsid protein from one serotype and a viral genome comprising different or heterologous serotype 5 ′ and 3 ′ inverted terminal repeats (ITRs). PseudorAAV can be expected to have cell surface binding properties of the capsid serotype and genetic properties consistent with the ITR serotype. Pseudotype rAAV can include IAVs from any serotype AAV, including AAV capsid proteins, including VP1, VP2, and VP3 capsid proteins, and any primate AAV serotype of AAV1-AAV12, provided that The capsid protein is assumed to be a serotype different from the ITR serotype. For pseudotyped rAAV, the 5 ′ and 3′ITR may be the same or different. Pseudo rAAV is generated using standard techniques described in the art.

  A “chimeric” rAAV vector includes an AAV vector containing a heterologous capsid protein, ie, the rAAV vector is chimeric with respect to its capsid proteins VP1, VP2, and VP3 such that VP1, VP2, and VP3 are not all of the same serotype of AAV It may be. Chimeric AAV as used herein is not limited to, for example, capsid proteins derived from AAV1 and AAV2, but capsid proteins VP1, VP2 and VP3 including these have different serotypes and other parvovirus capsid proteins. It is a mixture or includes other viral proteins or other proteins, such as AAV containing targeted delivery of AAV to a desired cell or tissue. As used herein, chimeric rAAV also includes rAAV comprising chimeric 5 ′ and 3 ′ ITRs. The present invention includes chimeric rAAV vectors comprising ITRs from different AAV serotypes, such as AAV1 and AAV2, and the chimeric rAAV can comprise synthetic sequences.

Vasodilators Vasodilation is the enlargement of blood vessels resulting from relaxation of smooth muscle in the vessel walls, arteries, arterioles, veins, and venules. As a result of vasodilation, vascular tolerance decreases and blood flow increases. Internal and external factors can induce vasodilation, and such factors are called vasodilators. There are two common mechanisms that cause vasodilation, a decrease in intracellular calcium and / or dephosphorylation of myosin light chain (MLC). These mechanisms are implemented by three general pathways: hyperpolarization-mediated, cAMP-mediated, or cGMP-mediated. Thus, vasodilators can exert their effects through one or more of these intermediate pathways. In one embodiment, the vasodilator is adenosine, histamine (or histamine inducer), alpha blocker, theobromine, papaverine, ethanol, tetrahydrocannabinol (THC), minoxidil, or nitric oxide (a nitric oxide augmenting substance). (Including but not limited to)). In one embodiment, only one vasodilator is administered. Another embodiment contemplates the use of one or more vasodilators together, sequentially, or a combination thereof. In a preferred embodiment, the vasodilator is a nitric oxide augmenting substance.

  Nitric oxide (NO) is a free radical molecule that can act as a short-lived chemical transmitter and can diffuse freely through the film. NO has various physiological effects. See generally Jeremy M. Berg, et al. (2006), Biochemistry, 6th Edition. W. H. Freeman and Company. For example, it is known to cause vasodilation by modulating smooth muscle contractility after systemic or local delivery. In the central nervous system, NO can affect synaptic transmission and stimulate learning and memory abilities. As another example, NO can induce platelet aggregation in plasma. Because of its lipophilicity, nitric oxide can diffuse from its originating cell to other neighboring cells, resulting in a signaling mechanism. In the coronary arteries, NO activates cytoplasmic guanylate cyclase, which can stimulate cyclic guanosine monophosphate (cGMP) formation in vascular smooth muscle cells, resulting in vasodilation.

  Without being limited to any particular mechanism of action, it has been discovered that coronary circulation, or vasodilation of the arteries supplying blood to the heart, can increase the efficiency of introduction of the therapeutic agents described further below. That is, the efficiency of introduction of a therapeutic agent can be increased by administering a combination of an agent capable of inducing vasodilators or vasodilation into the coronary circulation, cardiac arteries, or systemically, and preferably a NO-enhancing agent. . As used herein, a “NO-enhancing substance” includes a combination of 2, 3, 4, 5 or more compounds unless otherwise indicated, and does not actually increase the amount of NO, but to NO. Compounds that mimic an increase in NO by activating receptors, such as NO agonists, can be included. In some embodiments, treatment can be performed before, at least in part, or after treatment with the primary agent. Thus, in some embodiments, NO can be used as an adjuvant to increase the efficacy, efficiency, or efficacy of the primary therapeutic agent. Some embodiments include a combination of 2, 3, 4, 5 or more NO enhancers.

  Increasing levels of NO in the coronary circulation, even temporarily, can be performed by various known techniques. As used herein, NO augmenting substances are limited to any of the following compounds or classes of compounds, or any combination of 2, 3, 4, 5 or more of the following compounds or compound classes: Including these. Agents that release NO under physiological conditions have long been used in the management of heart disease. These agents can include NO donors, NO releasing molecules, NO precursors by way of example only. For example, NO donors can include nitrates, such as glyceryl trinitrate, which can also be commonly referred to as “nitroglycerin”. Other examples of nitrates are isosorbide nitrate and isosorbide mononitrate. NO donors are Megson IL, Webb DJ, “Nitric oxide donor drugs: current status and future trends” Expert. Opin. Investig. Drugs, 2002 May; 11 (5), the entire contents of which are incorporated herein by reference. ): Other agents such as those described in 587-601 can also be included. NO-releasing molecules can also increase the level of NO in the coronary circulation or coronary arteries. For example, the NO-releasing molecule can include diazeniumdiolate or a NO-releasing non-steroidal anti-inflammatory drug (NO-NSAID). NO precursors such as L-arginine can also be used to increase the level of NO. Other nitric oxide enhancing substances that can be used include molecular nitric oxide, nicorandil, and nitric oxide synthase, sodium nitroprusside, and pentaerythritol tetranitrate (PETN). In addition, agents that increase the effects of NO are contemplated, such as but not limited to sildenafil, tadalafil, and vardenafil, including phosphodiesterase type 5 (PDE5) inhibitors.

  In some embodiments, the material used to increase the amount of nitric oxide in the coronary circulation includes a nitric oxide donor. In some embodiments, the nitric oxide donor comprises nitrate. In a preferred embodiment, the nitrate comprises glyceryl trinitrate. In some embodiments, the nitrate comprises an agent selected from the group consisting of pentaerythritol tetranitrate, isosorbide nitrate and isosorbide mononitrate. In some embodiments, the nitric oxide donor comprises sodium nitroprusside. In some embodiments, the substance used to increase the amount of nitric oxide in the coronary circulation comprises nitric oxide releasing molecules. In some embodiments, the nitric oxide releasing molecule comprises an agent selected from the group consisting of diazeniumdiolate and a nitric oxide releasing non-steroidal anti-inflammatory drug. In some embodiments, the substance used to increase the amount of nitric oxide in the coronary circulation comprises an agent selected from the group consisting of molecular nitric oxide, nicorandil, and nitric oxide synthase. . In some embodiments, the substance used to increase the amount of nitric oxide in the coronary circulation includes a nitric oxide precursor. In some embodiments, the nitric oxide precursor comprises L-arginine.

Administration of vasodilatory substances
One or more vasodilators include, but are not limited to, systemic, eg, oral administration including but not limited to sublingual and translingual administration, patches or ointments Administration can be by transdermal administration, or intravenous injection or infusion. In a preferred embodiment, the one or more vasodilators can be administered by intracoronary injection or infusion. In another preferred embodiment, the one or more vasodilators can be administered by intravenous injection or infusion. The following sections further describe these delivery forms.

  The coronary circulation provides a blood supply to the heart tissue. Intracoronary administration is performed by injection or infusion into one or more blood vessels of the heart's coronary circulation that beats in vivo. There are several coronary arteries. Typically, the four main coronary arteries, the left main and right coronary arteries, the left anterior descending artery, and the left circumflex artery provide oxygen-rich blood to the heart through circulation through the heart tissue. Injection or infusion of one or a combination of these arteries is contemplated, such as injection or infusion into the left and right coronary arteries. In one embodiment, two thirds of the total amount of one or more vasodilators, including but not limited to one or more NO enhancers, is administered to one blood vessel of the heart. , 1/3 is administered to another blood vessel in the heart. In another embodiment, a portion of the total volume or amount of vasodilator that is injected or infused into three or more coronary vessels (e.g., 3, 4, 5 or more) and administered per vessel is optionally Can be adjusted. Preferred embodiments utilize antegrade, epicardial injection, or infusion of the left and right main coronary arteries. Coronary retrograde injections or infusions or a combination of one or more antegrade and retrograde coronary arteries or veins are also contemplated.

  Injection or infusion of one or more vasodilators into the coronary artery blood vessel (s), including but not limited to the NO enhancer (s), is required Depending on the standard guidewire, catheter and infusion pump. In a preferred embodiment, an injection or infusion catheter is directed to the coronary artery under fluoroscopy via the femoral artery. As used herein, “coronary vascular”, “coronary blood vessel” or “cardiac blood vessel” includes transplantation into a coronary blood vessel, eg, a graft resulting from a bypass surgical transplant. As used herein, “epicardium” refers to a blood vessel located in the outer portion of the heart, such as the left or right coronary artery.

  The amount of vasodilatory substance administered to a subject depends on the size of the subject and the route of administration. In a preferred embodiment, one or more vasodilators, including but not limited to one or more NO enhancers, are administered for about 5 minutes prior to administration of the viral vector or other therapeutic agent. Less than directly into the coronary arteries (typically 0.1-2 mL volume, less than 1 minute) as a single bolus injection. In some embodiments, the vasodilator (s) including the NO enhancer (s) is about, at least, at least about, long before administration of the viral vector or therapeutic agent. Or about 0.5, 1, 2, 3, 4, 5, 7, 10, 12, 15, 20, 25, or 30 minutes, 1, 2, 3 or more hours, or as described above Administer locally or systemically by a time that is in the range defined by any two of the values, preferably by injection or infusion. In preferred embodiments, this range is 0.5 to 10 minutes. More preferably, the vasodilator (s) or NO enhancer (s) is administered immediately prior to administration of the viral vector or therapeutic agent, preferably by a single bolus injection. In some embodiments, where the vasodilator (s) is administered by infusion, the administration of the viral vector or therapeutic agent is about, at least, at least about, after completion of the vasodilator infusion. , At least about 0.5, 1, 2, 3, 4, 5, 7, 10, 12, 15, 20, 25, or 30 minutes, 1, 2, 3 or more hours, or at least Starts with a time that is a range defined by any two of the previous values.

  In some embodiments, the vasodilator (s) or NO enhancer (s) is injected or infused prior to the viral vector or therapeutic agent described herein, A second dose of the same or different (one or more) vasodilatory substances or (one or more) NO-enhancing substances, preferably for at least 3 minutes, more preferably from about 4 to about 10 Administered in minutes or simultaneously with the viral vector or therapeutic agent as described in more detail below. In other embodiments, without prior treatment with the vasodilator (s) or NO enhancer (s), the vasodilator (s) It is administered at the same time as a viral vector or therapeutic agent to be administered as described herein. In some embodiments, the co-administration is the administration of a vasodilator in the same solution as the therapeutic agent. In other embodiments, the co-administration is by different routes of administration for the vasodilator and the therapeutic agent (eg, intravenous and intracoronary, respectively).

  In some embodiments, one or more vasodilators are administered after the viral vector or therapeutic agent, including but not limited to one or more NO enhancers. This post-administration may be in addition to pre-treatment and / or co-administration with the therapeutic agent, and may be the same or different (one or more) vasodilatory as administered in pre-treatment and / or co-administration It may be a substance. In some embodiments, the vasodilator (s) or NO enhancer (s) may be about, at least, at least about, longer than after administration of the viral vector or therapeutic agent. , Or at most about 0.5, 1, 2, 3, 4, 5, 7, 10, 12, 15, 20, 25, or 30 minutes, 1, 2, 3 or more hours, or the above values Administration is by a time that is in the range defined by any two, preferably by injection or infusion. In a preferred embodiment, this range is 0.5-10 minutes after administration of the viral vector.

  In a preferred embodiment, the NO enhancer is nitroglycerin and the total amount of nitroglycerin administered by intracoronary injection or infusion at one or more of the doses described herein is from about 50 μg to about 500 μg, or more. Preferably, it is about 100 μg to about 150 μg. Contemplated total amount or administration of nitroglycerin, or other (one or more) vasodilators or (one or more) NO augmenting substances, or combinations of substances, administered by intracoronary injection or infusion Per unit is about, at least, at least about, at most, or at most about 1, 2, 3, 4, 5, 7, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80 , 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 μg, or a range defined by any two of the aforementioned values It is. This amount may be the amount administered to one coronary artery or all coronary arteries that will be injected or infused, or any combination thereof, with or after the therapeutic agent, or the total amount administered. It's okay. Those skilled in the art understand that the dose of vasodilator used for intracoronary injection or infusion is related to organ size and not necessarily the total body weight of the subject. In one embodiment, an initial intracoronary injection of 50 μg nitroglycerin is given prior to infusion of the viral vector, and a second amount of 100 μg nitroglycerin is preferably about at least 3 minutes, more preferably about 4 minutes. Inject with viral vector for up to 10 minutes.

  In some embodiments, the total dose of nitroglycerin administered systemically by intravenous injection or infusion is preferably about 200 μg to about 4000 μg, more preferably about 500 μg to about 2500 μg. The contemplated total dose of nitroglycerin administered by systemic injection or infusion is about, at least, at least about, at most, or at most about 1, 2, 3, 4, 5, 7, 10, 12, 15, 20 , 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1250, 1500 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 μg, or a range defined by any two of the aforementioned values. In some embodiments, if nitroglycerin is administered intravenously at 5 μg / min or about 5 μg / min and increased by 5 μg / min every 3-5 minutes up to 20 μg / min, the dose is 200 μg if there is no response at 20 μg / min. It can be increased by 10 μg / min every 3-5 minutes up to / min. Contemplated dose rates of nitroglycerin administered by systemic injection or infusion are about, at least, at least about, at most, or at most about 1, 2, 3, 4, 5, 7, 10, 12, 15, 20 , 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, or 400 μg / min or in a range defined by any two of the aforementioned values is there. The total time of infusion of the vasodilator (s) or NO augmenting agent (s) is about, at least, at least about, at most, or at most about 5, 7, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, or 600 minutes, or a range defined by any two of the aforementioned values. In some embodiments, the IV infusion begins prior to administration of the viral vector or therapeutic agent and continues during the administration.

  In some embodiments, the total dose of nitroglycerin administered systemically by oral means is preferably about 5 mg to about 105 mg, more preferably about 10 mg to about 80 mg. In another embodiment, the preferred dose is about 15 mg to about 80 mg. Contemplated total doses of nitroglycerin administered orally are about, at least, at least about, at most, or at most about 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 7, 10, 12, 15 20, 30, 40, 50, 60, 70, 80, 90, 100, or 125 mg, or a range defined by any two of the aforementioned values.

  In another embodiment, the amount of nitroglycerin given systemically by sublingual administration is about, at least, at least about, at most, or at most about 36, 54, 72, 90, 108, 126, 144, 162, 180. , 198, 216, 234, 252, 270, 288, 306, 324, 342, or 360 mg, or a range defined by any two of the aforementioned values. In some embodiments, nitroglycerin is administered sublingually, wherein the dose is about, at least, at least about, at most, or at most about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5. 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1 mg, or a range defined by any two of the aforementioned values. The sublingual dose is about, at least, at least about, at most, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes or any two of the aforementioned values Administer at intervals that are in the range defined by In a preferred embodiment, sublingual administration of nitroglycerin provides about 0.2 to about 0.6 mg every 5 minutes for a maximum of 3 doses every 15 minutes. In another embodiment, nitroglycerin can be given translingually by spray, drop, or mist. 1-2 sprays in the mouth can be given every 3-5 minutes for up to 3 doses in 15 minutes.

  In some embodiments, systemic administration of nitroglycerin can be performed by transdermal delivery using a transdermal patch. The total dose of nitroglycerin administered systemically via a transdermal patch is preferably about 2.4 mg to about 15.6 mg, more preferably about 4.8 mg to about 9.6 mg. Contemplated total doses of nitroglycerin administered via a transdermal patch are about, at least, at least about, at most, or at most about 2.4, 3.6, 4.8, 6, 7.2, 8.4, 9.6, 10.8, 12, 13.2, 14.4, or 15.6 mg, or a range defined by any two of the aforementioned values. In some embodiments, the nitroglycerin is administered via a transdermal patch, and the dose is about, at least, at least about, at most, or at most about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, or 0.65 mg, or a range defined by any two of the aforementioned values. The transdermal patch dose is about, at least, at least about, at most, or at most about every 15, 30, 45, 60, 75, or 90 minutes, or in a range defined by any two of the aforementioned values Administer at certain intervals. In another embodiment, contemplated dose rates of nitroglycerin administered via a transdermal patch are about, at least, at least about, at most, or at most about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1 mg / hour, or a range defined by any two of the aforementioned values. In a preferred embodiment, nitroglycerin is given transdermally at an initial dose of a dose of about 0.2-0.4 mg / hour, up to 0.4-0.8 mg / hour. By using a 12-14 hour patch-on period and a patch-off period of about 10-12 hours, tolerance is minimized.

  In another embodiment, systemic administration of nitroglycerin can be performed by transdermal delivery by topical application of an ointment. Contemplated total doses of nitroglycerin administered by topical ointment are about 0.1%, 0.15%, 0.2% at least about 1/2 "square inch when elevated, and 1/2" square inch after 6 hours, 0.25%, 0.3%, 0.4%, 1%, or 2% nitroglycerin concentration, or a range defined by any two of the aforementioned values. In a preferred embodiment, the ointment concentration is 0.2%. The dose can be doubled as needed.

  Some embodiments contemplate systemic delivery of nitroglycerin through the skin by subcutaneous injection or infusion. In some embodiments, the total dose of nitroglycerin administered subcutaneously is preferably about 5 mg to about 105 mg, more preferably about 10 to about 80 mg. In another embodiment, the preferred dose is about 15 mg to about 80 mg. Contemplated total doses of nitroglycerin administered subcutaneously are about, at least, at least about, at most, or at most about 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 7, 10, 12, 15 20, 30, 40, 50, 60, 70, 80, 90, 100, or 125 mg, or a range defined by any two of the aforementioned values.

  In a preferred embodiment, the vasodilator (or agents) or NO enhancer (or agents) or combination of substances are added in an amount sufficient to increase vasodilation or vascular permeability, any other Administer without vasodilators or vascular penetration enhancers. In some embodiments, the NO augmenting substance or substance, preferably a combination of nitroglycerin, is the only vasodilator or vascular penetration enhancer that is administered before, during and / or after administration of the viral vector or therapeutic agent. It is. In some embodiments, no vasodilator or vascular penetration enhancer is administered prior to, during and / or after administration of the viral vector or therapeutic agent other than the NO enhancer described herein. In one embodiment, the NO enhancer described herein, preferably other than nitroglycerin, sufficient to increase uptake of the viral vector or therapeutic agent before, during and / or after administration of the viral vector or therapeutic agent The subject is not treated with any amount of any vasodilator or permeation enhancer. Vasodilators or permeation enhancers that are preferably excluded from administration in some embodiments include, but are not limited to, vascular endothelial growth factor (VEGF), adenosine, and calcium.

  In some embodiments, the amount of one or more vasodilators is an increase in transfection or a pharmaceutically or therapeutically effective amount, which increases the efficiency of transduction of the viral vector or therapeutic agent. Enough to do. Increased transfection can be measured directly by examining transfection efficiency, or measures other indicators of successful transfection, such as an improvement in one or more symptoms or outcomes discussed herein. Can be measured indirectly. An increase amount is an amount that improves the index as compared to the same index when not administering the vasodilator (s) or NO enhancer (s).

Administration of Therapeutic Agents In a preferred embodiment of the invention, a therapeutic agent as described in more detail below, for example a polynucleotide / viral vector, is pulsated in the coronary circulation of the heart that beats in vivo for at least 3 minutes in a particular blood vessel. Administered to the subject by injection. In large animal models of human heart and cardiovascular disease, Applicants have indicated that administration of a relatively long infusion time of a viral vector is more than a bolus injection of the same amount of viral vector or a short (eg, 1 minute or less) infusion time. It has been unexpectedly found to be effective and provide excellent gene transfer efficiency into heart tissue. Improved efficiency of infusion is due to higher copy number of transgene per cell, increased expression of transgene at the mRNA and / or protein level per cell or in tissue and / or transfection compared to injection It can be measured as a high percentage of cells of a specific tissue, such as cardiomyocytes. In another embodiment, clinical or functional measures can be used to demonstrate transfection efficiency from relatively long infusion times. Such clinical and functional assessment is further described herein.

  Applicants have shown that this method provides a successful treatment of large animal models of human cardiovascular disease. In addition, Applicants have used a relatively long infusion time to separate the coronary circulation from the systemic circulation, or otherwise recirculate the therapeutic agent, or to increase the pressure in the coronary circulation or the therapeutic agent. It has been discovered that there is no need to artificially limit coronary venous circulation as a means for increasing residence time. There is also no need to cool the heart, stop the heart, or remove the heart from the animal for perfusion. Instead, Applicants' procedures can be performed in a standard catheterization laboratory setting using existing dosing catheters. Accordingly, Applicants have discovered a simple, practical and effective means of using gene therapy to treat cardiovascular disease in large animals such as humans.

  In a preferred embodiment of the invention, the therapeutic agent is administered to the subject by infusion into the coronary vasculature. The coronary circulation provides a blood supply to the heart tissue. There are several coronary arteries. Usually, the four main coronary arteries; the left main and right coronary arteries, the left anterior descending coronary artery, and the left circumflex branch provide oxygen-containing blood to the heart for distribution through the heart tissue. Infusion of one or a combination of these arteries, for example, left and right coronary arteries is contemplated. A preferred embodiment utilizes antegrade epicardial infusion of the left and right main coronary arteries. Coronary retrograde injection or a combination of one or more antegrade and retrograde coronary arteries or veins is also contemplated. Coronary vascular infusion is performed using a standard guidewire, catheter and infusion pump. In a preferred embodiment, the infusion catheter is directed to the coronary artery under fluoroscopy via the femoral artery. As used herein, “coronary vascular”, “coronary blood vessel” or “cardiac blood vessel” includes transplantation into a coronary blood vessel, eg, a graft resulting from a bypass surgical transplant. As used herein, “epicardium” refers to a blood vessel located in the outer portion of the heart, such as the left or right coronary artery.

  Once the infusion catheter is in place in the target coronary artery vessel, the therapeutic agent is infused into the vessel, preferably by a programmable infusion pump. The amount of time required to inject the therapeutic agent is an important factor in obtaining effective and excellent gene transfer efficiency. Applicants have determined that infusion times of at least about 3 minutes into a particular blood vessel are more effective than bolus injections or even shorter infusion times. Preferably, the infusion time is at least about 8 minutes, more preferably at least about 10 minutes, although an infusion time of at least about 15 minutes is contemplated. Applicants have noted that the infusion time is at least about 1, at least about, at most, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, It is also contemplated that it is 14, 15, 16, 17, 18, 19, or 20 minutes, or within the range defined by any two of these values.

  Since infusion typically involves the use of a catheter and a connecting tube having a dead volume, the infusion device is often filled with a carrier solution that does not contain any therapeutic agent, such as blood from the subject. Thus, the therapeutic agent is not immediately administered to the coronary circulation when the infusion pump is activated. Similarly, when a syringe containing a therapeutic agent is empty, a certain amount of therapeutic agent typically remains in the dead volume of the connecting tube and catheter. Immediately after infusion of the therapeutic agent, flush the dead volume with an appropriate solution. For dead volume movement within the infusion device, the time period during which the therapeutic agent is actually delivered to the coronary circulation is the previously mentioned “infusion time”. For example, if 3 mL of therapeutic agent is filled into an infusion device with a 3 mL dead volume and the infusion rate is 1 mL / min, the time required to inject the therapeutic agent into the coronary circulation is only 3 minutes and 3 mL The total time required to administer 3 mL of therapeutic agent and 3 mL dead volume is 6 minutes. In some embodiments, the catheter and any connecting tubing are filled with a therapeutic agent so that dead volume is not an issue. Similarly, it was possible to deliver an effective amount of therapeutic agent without having to flush the tube. However, this results in residual therapeutic agent in the tube and wastes the therapeutic agent.

  Applicants have at least about, at least, at least about, at most, or at most about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6. 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 It is contemplated to inject the therapeutic agent at a flow rate that is, 9.0, 9.5, or 10.0 mL / min, or in the range defined by any two of these values. Preferably, the flow rate is between about 0.2 mL / min and about 6.0 mL / min, more preferably between about 0.2 mL / min and about 2.5 mL / min, more preferably about 0.2 mL / min and about 2.0 mL / min. Between. One skilled in the art should understand that delivery of therapeutic agents without an infusion pump is possible, however, more accurate flow rates and uniform delivery are possible through the use of an infusion pump.

The total amount of viral or DNase resistant particles (DRP) delivered by injection to provide an effective amount is preferably between 1 × 10 14 and about 1 × 10 11 , more preferably about 3 × 10 12 and 1 ×. Between 10 12 and more preferably about 3 × 10 12 . However, Applicants have noted that the total amount of viral particles or DRP is about, at least, at least about, at most, or at most about 1 × 10 14 , 9 × 10 13 , 8 × 10 13 , 7 × 10 13 , 6 ×. 10 13 , 5 × 10 13 , 4 × 10 13 , 3 × 10 13 , 2 × 10 13 , 1 × 10 13 , 9 × 10 12 , 8 × 10 12 , 7 × 10 12 , 6 × 10 12 , 5 × 10 12 , 4 × 10 12 , 3 × 10 12 , 2 × 10 12 , 1 × 10 12 , 9 × 10 11 , 8 × 10 11 , 7 × 10 11 , 6 × 10 11 , 5 × 10 11 , 4 × 10 11 , 3 × 10 11 , 2 × 10 11 , 1 × 10 11 , 9 × 10 10 , 8 × 10 10 , 7 × 10 10 , 6 × 10 10 , 5 × 10 10 , 4 × 10 10 , 3 × 10 10 , 2 × 10 10 , 1 × 10 10 , 9 × 10 9 , 8 × 10 9 , 7 × 10 9 , 6 × 10 9 , 5 × 10 9 , 4 × 10 9 , 3 × 10 9 , 2 × It is also contemplated to be in the range defined by 10 9 , 1 × 10 9 , or any two of these values.

The number of DRP to infuse for a given time is a function of the concentration of the solution to be infused and the flow rate. The rate of DRP or virus particle injection is preferably between about 1 × 10 8 / min and about 1 × 10 14 / min, more preferably between about 5 × 10 10 / min and about 5 × 10 12 / min, More preferably between about 3 × 10 10 / min and about 1 × 10 12 / min, more preferably between about 6 × 10 10 / min and about 4 × 10 11 / min. In a preferred embodiment, the rate of DRP or virus particle injection is 1 × 10 11 / min, and in other preferred embodiments it is 1.25 × 10 11 / min.

  In one embodiment, the therapeutic agent is administered to a single blood vessel in the heart. In other embodiments, two thirds of the total volume of the therapeutic agent is delivered to one blood vessel of the heart and one third is administered to the other blood vessels of the heart. In other embodiments, three or more coronary vessels are infused (e.g., 3, 4, 5 or more), and the percentage of the total infused volume containing the therapeutic agent administered per vessel is adjusted as needed. be able to. The purpose of the infusion is to provide extensive, homogeneous left ventricular myocardial exposure to AAV2 / 1 / SERCA2a by antegrade, epicardial coronary infusion. Although there are numerous infusion scenarios based on concomitantization patterns, occlusive disease, and anatomical variations (e.g., anatomy after bypass surgery), the clinician's goal is 1 / A of AAV2 / 1 / SERCA2a 3 is delivered anterolaterally, 1/3 is delivered posteriorly and 1/3 is delivered to the lower / lower lateral myocardium. In order to perform homogeneous delivery to the perfused myocardium, anatomy is defined by coronary artery and bypass grafts. Furthermore, those skilled in the art should understand that sheep and pigs are accepted animal models for human cardiovascular testing, while sheep and pigs are 90% left dominant. In comparison, up to about 10% of the human population is left dominant and the remaining 90% is right or mutual dominant (Vlodaver Z. et al. Coronary Heart Disease: Clinical, Angiographic, and Pathologic Pofiles. Spinger-Verlag , New York. 1976). One pathology system suggests that 71% of patients are right dominant, 17% are mutually dominant, and 12% are left dominant (McAlpine W. Heart and Coronary Ateries. Spinger Verlag, 1975) . Thus, in order to perform similar perfusion of the left ventricle in human versus pig / sheep, the optimal infusion scenario may be different.

  A division of 1/3 and 2/3 of the solution volume is preferred for two vessels, however, the proportion of infusion injected into a particular vessel is about, at least, at least about, more than the total volume, or At most about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80%, or within the range defined by any two of these values It can be a certain amount. The total amount of solution containing the therapeutic agent will vary depending on the size of the animal being treated. For human subjects, a total therapeutic amount of 60 mL is preferred. However, the total amount of therapeutic agent is about, at least, at least about, at most, or at most about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 mL, or any of these values Or an amount in the range defined by the two.

  The therapeutic agent described herein may be a solution, preferably a pharmaceutical composition, suitable for direct administration into the coronary circulation. The components of an acceptable pharmaceutical composition are known to those skilled in the art and can include elements such as buffers and suitable carriers. In other embodiments, the pharmaceutical composition comprising a therapeutic agent, eg, a viral vector, and more preferably an AAV2 / 1 / SERCA2a vector is part of the kit. In some embodiments, the kit includes a stock solution of the therapeutic agent and a solution for diluting the stock solution. Instructions regarding the administration of the viral vector, preferably by direct injection into the coronary circulation as described in any of the embodiments disclosed herein, are also included in the kit. The therapeutic agent (s) and vasodilator (s) including but not limited to the NO enhancer (s) described herein treat the diseases disclosed herein. Can be used in the manufacture of a medicament for administration in which case the medicament is administered according to or in accordance with any of the methods disclosed herein.

Methods of Polynucleotide Delivery One aspect of the present invention contemplates the introduction of therapeutic polynucleotides into cells. Such introduction can utilize viral or non-viral gene transfer methods. This section provides a discussion of methods and compositions for gene or nucleic acid transfer, including the introduction of antisense, interference, small interfering sequences.

  In one embodiment, a therapeutically significant polynucleotide is incorporated into a viral vector to mediate introduction into the cell. Other expression constructs encoding other therapeutic agents described herein are also viruses that use infectious viral particles, for example, by transformation with an adeno-associated virus (AAV) or AAV molecular variant of the invention. It can be introduced by transduction. Alternatively, retroviruses, bovine papillomaviruses, adenovirus vectors, lentivirus vectors, vaccinia viruses, polyoma viruses, or infectious viruses engineered to express can be used. Similarly, use non-viral methods, including but not limited to direct delivery of DNA, such as by perfusion, naked DNA transfection, liposome-mediated transfection, encapsulation, and receptor-mediated endocytosis be able to. These techniques are well known to those skilled in the art and the details are not at the heart of the present invention and therefore need not be detailed in detail herein. However, in a preferred example, viral vectors are used to transduce heart cells to deliver therapeutically significant polynucleotides to the cells. Viruses can enter inside cells by specific means such as receptor-mediated endocytosis or by non-specific means such as pinocytosis.

Adeno-associated virus vectors A preferred embodiment of the present invention utilizes purified, non-replicating, pseudotyped recombinant adeno-associated virus (rAAV) particles. Adeno-associated virus (AAV) is a parvovirus belonging to the genus Dependovirus. They are small, non-enveloped, single-stranded DNA viruses that require a helper virus to replicate. Superinfection with a helper virus (eg, adenovirus, herpes virus, or vaccinia virus) is necessary to form a functionally complete AAV virion. In vitro, in the absence of superinfection with helper virus, AAV establishes a latent state during which the viral genome exists in an episomal form, but no infectious virions are produced. Subsequent infection with helper virus can "rescue" the genome, replicate it and package it into the viral capsid, thereby reconstructing the infectious virion. Recent data indicate that both wild-type and recombinant AAV are predominantly present as large episomal concatamers in vivo.

  AAV is not associated with any known human disease, is generally not considered pathogenic, and does not appear to alter the host cell's physiological properties upon integration. AAV can infect a wide range of host cells, including non-dividing cells, and can infect cells from different species. In contrast to some vectors that are readily removed or inactivated by both cellular and humoral responses, AAV vectors have shown sustained expression in various tissues in vivo. The persistence of recombinant AAV vectors in non-dividing cells in vivo may be due to the lack of prototypic AAV viral genes and the ability of the vectors to form episomal concatamers.

  Adeno-associated virus (AAV) is an attractive vector system for use in cell transduction, because it has a high frequency of persistence as an episomal concatamer, can infect non-dividing cells, and therefore sucks This is because it is useful for delivery of genes to animal cells such as tissue culture and in vivo. Studies demonstrating the use of AAV in gene delivery include Flotte et el., Proc. Natl. Acad. Sci. USA, 1993; 90: 10613-17 and Walsh et al., J Clin. Invest., 1994; 94 : 1440 ~ 48 Recombinant AAV vectors have been successfully used for in vitro and in vivo transduction of marker genes and genes associated with human disease (eg Walsh et el., J. Clin. Invest. 1994; 94: 1440-48). AAV has a broad range of host infections. Details regarding the generation and use of rAAV vectors are described in US Pat. No. 5,139,941 and / or US Pat. No. 4,797,368, each incorporated herein by reference.

  Typically, recombinant AAV (rAAV) viruses contain a plasmid containing the gene of interest adjacent to two AAV terminal repeats and / or an expression plasmid containing a wild type AAV coding sequence without terminal repeats, e.g., pIM45. Produced by co-transfection. Cells are further infected and / or transfected with adenovirus and / or plasmids carrying adenoviral genes required for AAV helper function. RAAV virus stocks made in this manner are contaminated with adenovirus and must be physically separated from rAAV particles (eg, by cesium chloride density gradient centrifugation or column chromatography). Alternatively, an adenoviral vector containing an AAV coding region and / or a cell line containing an AAV coding region and / or part or all of an adenoviral helper gene can be used. Cell lines with rAAV DNA as the integrated provirus can also be used.

A number of serotypes of AAV exist in nature, and at least 12 serotypes (AAV1-AAV12) are currently known. In addition, chimeric variants have been generated by directed evolution (DNA shuffling) technology (see Li et al.). Despite the high degree of homology, different serotypes are directed against different tissues. The receptor for AAV1 is not known, however, AAV1 is known to transduce skeletal and cardiac muscle more efficiently than AAV2. Because most studies have been performed using pseudotyped vectors in which the AAV2 ITR and adjacent vector DNA are packaged into capsids of other serotypes, biological differences are related to the capsid rather than the genome. It is clear that there is. Recent evidence indicates that the DNA expression cassette packaged in the AAV1 capsid is at least 1 log 10 more effective than the cassette packaged in the AAV2 capsid in transducing cardiomyocytes.

Engineered rAAV Vectors In one embodiment, AAV vectors can be engineered to reduce neutralizing antibody (NAb) titers and / or cross-reactivity. The cross-reactivity of Nab with engineered or chimeric vectors is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, or 99% greater than that of wild-type vectors Preferably it is low. More preferably, cross-reactivity is essentially absent. Cross-reactivity and / or reduction of Nab titer can be performed by engineering modifications of the AAV capsid protein to create chimeric and / or modified rAAV vectors. DNA shuffling (family or single gene), all of which are incorporated herein by reference, Li et al., Crameri, A et al. (1998). from diverse species accelerates directed evolution.Nature 391: 288-291 and Stemmer, WP (1994) .See Rapid evolution of a protein in vitro by DNA shuffling.Nature 370: 389-391.) Mutation, Variant PCR (Moore, GL, Maranas, CD, 2000, "Modeling DNA mutation and recombination for directed evolution experiments." J. Theor. Biol. 205, 483-503, incorporated herein by reference. ), The creation of chimeras, or combinations thereof, such as an alternating extension process that incorporates DNA shuffling and mutated PCR techniques (Mesheshri, N et al. (Incorporated herein by reference in its entirety). 2006) .``Directed evolution of adenoassociated virus yields enhanced ge ne delivery vectors. "Nat Biotechnol 24: 198-204). Several methods are known in the art for engineering modification of genes, including, but not limited to, capsid genes. It has been. After engineering the capsid protein, the rAAV vector is assayed for the properties desired for selection or screening. For example, selection and / or screening can be used to isolate rAAV clones that incorporate properties that result in reduced reactivity or cross-reactivity to Nab. As described in Li et al., Modified rAAV vectors were generated after using DNA shuffling techniques on the gene responsible for the AAV capsid protein code (ie, the cap gene). The engineered rAAV clones generated by these authors were generated from multiple serotype combinations and thus contained genomic fragments representing various parental serotypes. In order to evaluate the immunological profile of the modified rAAV vector, it was subjected to a series of cross-reactivity studies. In these studies, antisera were collected from mice immunized with the specific AAV serotype from which the engineered rAAV vector was derived (ie, the parental serotype). The assay in the Li et al. Study evaluated the extent of cross-reaction of engineered rAAV vectors with antisera generated from mice immunized with Nab titers and AAV parental serotypes. The results showed that antisera from three of the four parental serotypes did not cross-react with engineered rAAV clones, while the remaining samples showed 25-fold lower Nab titers.

  In another embodiment, AAV vectors can be engineered to increase transduction efficiency and / or specificity. Increased transduction efficiency and / or specificity can be performed by engineering the AAV capsid protein to create chimeric and / or modified rAAV vectors. Limited to that combination, such as DNA shuffling (family or single gene), site-directed mutagenesis, mutated PCR, chimera creation, or alternating extension process, a method that incorporates DNA shuffling and mutated PCR techniques A number of methods are known in the art for engineering genes, including but not limited to these. After engineering the capsid protein, the rAAV vector is assayed for the desired properties to assay. For example, selection and / or screening can be used to isolate rAAV variants that have gained increased efficiency and / or specificity for the target tissue (s) or cells.

Therapeutic Effects In a preferred embodiment, infusion of the therapeutic agents disclosed herein is used to obtain a therapeutic effect in patients suffering from heart disease. Individuals to be treated can be monitored for clinical features associated with cardiac disorders to determine if a therapeutic effect has been achieved. For example, a subject can be monitored for adverse effects and reduced symptoms associated with cardiovascular disease. For example, after treating congestive heart failure in a subject using the methods disclosed herein, increased lateral ventricular shortening rate, increased myocardial contractility at the cellular and intact animal level, reversal of cardiac remodeling, Subjects can be evaluated for improvements in several parameters, including but not limited to normalization of cytosolic calcium at abnormally high diastolic levels. Other clinical and cardiac parameters that can be monitored in subjects treated using the techniques of the present invention include survival, cardiac metabolism, myocardial contractility, heart rate, ventricular function (e.g., left ventricular end diastolic pressure (LVEDP) Left ventricular end systolic pressure (LVSP)), Ca 2+ metabolism (eg, intracellular Ca 2+ concentration, peak or resting state [Ca 2+ ], SR Ca 2+ ATPase activity, phosphorylation state of phospholamban) Force generation, relaxation, force contraction frequency relationship, cardiac cell survival or apoptosis or ion channel activity (e.g. sodium and calcium exchange, sodium channel activity, calcium channel activity, sodium potassium ATPase pump activity), myosin heavy chain, BNP And NT-proBNP, troponin I, troponin C, troponin T, CK-MB, tropomyosin, actin, myosin light chain kinase, myosin light chain 1, myosin light 2 or myosin light chain 3, IGF-1 receptor, PI3 kinase, AKT kinase, sodium-calcium exchanger, calcium channels (L and T), calsequestrin or calreticulin, but only Not limited. Evaluation can be performed before, after, and during treatment. Other measures of heart disease that can be monitored include shortening rate, cardiac output, ejection fraction, Tau, reverse flow rate, short-term hospitalization, quality of life, and treadmill time, during a 6-minute walk test The distance walked to and the maximum oxygen consumption (VO 2 max). In one embodiment, the patient is monitored using molecular biology techniques known in the art,
RAAV vector DNA, RNA, and / or protein present in cells and / or tissues can be measured. In some embodiments, the transgene copy number per cell, the expression of the transgene at the mRNA and / or protein level per cell or in the tissue, and / or cells of a particular tissue to be transfected, such as cardiomyocytes Can be evaluated.

  In a preferred embodiment, administration of a vasodilator, preferably a NO enhancer as described herein, more preferably nitroglycerin, increases the efficiency of introduction of the therapeutic agent. In some embodiments, administration of a vasodilator or NO enhancer improves the therapeutic effect obtained by administration of the therapeutic agent alone, where the therapeutic effect is monitored as described herein. In some embodiments, administration of a vasodilator or NO enhancer results in improved efficacy of the therapeutic agent such that the same level of therapeutic effect can be obtained with less therapeutic agent. Improved efficacy may result in less therapeutic agent required in a single dose, or fewer doses of therapeutic agent over time. In some embodiments, administration of a vasodilator improves the therapeutic effect obtained by administration of the therapeutic agent alone by extending the duration of the therapeutic effect. In some embodiments, the improved introduction efficiency, therapeutic effect, or efficacy of the therapeutic agent is about, at least, at least about 5, 10, when compared to values obtained without administering a vasodilator. 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 125, 150, 175, or 200%, or 2, 3, 4, 5 , 7, 10, 15, or 20 times or a range defined by any two of the aforementioned values. For example, if an increase in ejection fraction is observed to last 3 months after treatment without vasodilators, a 50% increase in treatment effect after treatment with vasodilators will last for 4.5 months This will increase the rate.

  The methods disclosed herein and the disclosed therapeutic agents can be combined with existing treatments for heart disease, such as drugs or surgical interventions, to provide increased therapeutic effects compared to existing treatments alone. Increased therapeutic effect is, for example, extended time period between worsening signs or symptoms of disease compared to average or typical time period for existing treatment regimens, or compared to average or typical time for standard treatment alone And can be demonstrated by the increased time required before further treatment is needed.

Other embodiments contemplated herein are kits comprising a therapeutic agent, such as a viral vector, and more preferably a container of an AAV2 / 1 / SERCA2a vector, and a container of one or more vasodilators. . In some embodiments, the kit contains a stock quantity of the therapeutic agent and a carrier solution for dissolving or diluting the stock quantity. In some embodiments, the kit dissolves or dissolves a stock quantity of vasodilator (s), including but not limited to (one or more) NO augmenting substances, and a stock quantity. Contains a carrier solution for dilution. In some embodiments, the kit contains a mixture of a container and a therapeutic agent and an amount of vasodilator (s). Stock quantities of therapeutic agents and / or vasodilator (s) are in a carrier solution, in a dry form that requires dissolution or mixing in a concentrated solution that requires dilution, or no additional preparation It may be in a form that is in a state of being administered to a patient. In some embodiments, the kit includes one or more intravascular infusion or injection catheters for intracoronary administration of the vasodilator and / or therapeutic agent. In some embodiments, the kit includes one or more devices for administering the compounds in the kit via a catheter, such as a syringe. The kit can also include instructions for administration of the viral vectors and / or therapeutic agents described in any of the embodiments disclosed herein, preferably by direct injection into the coronary circulation.

  In the following non-limiting examples, embodiments of the invention will now be further described. All references disclosed herein, including patents and non-patent literature, are hereby incorporated by reference in their entirety, particularly with respect to their disclosures referred to herein.

[Example 1]
A 30-day, single-dose, tissue distribution study of direct intracoronary infusion of AAV1 / SERCA2a (MYDICAR®) in normal Gottingen piglets
I. Protocol overview

Administration Method Administration on day 0 by the direct intracoronary infusion procedure described below. Animals from groups 1-3 were administered a total volume of 12 mL of AAV1 / SERCA2a solution, which was infused at a constant rate of 1.2 mL / min for 10 minutes. Animals from groups 3 and 4 were given nitroglycerin as a bolus intracoronary injection just prior to administration of AAV1 / SERCA2a. Animals from group 3 received a total volume of 12 mL of AAV1 / SERCA2a and nitroglycerin solution, which was infused at a constant rate of 1.2 mL / min for 10 minutes. Animals from group 4 received a total volume of 12 mL normal saline, which was infused at a constant rate of 1.2 mL / min for 10 minutes. The direct infusion system consists of standard (commercial) components, including a conventional guide sheath, 0.014 "guidewire, 5F infusion (guide) catheter and two programmable syringe pumps. The infusion procedure began with the introduction of a conventional guide sheath using common carotid or femoral artery techniques, such as a coronary artery infusion catheter (eg Cordis Vista Brite Tip Guiding Catheter or similar model suitable for intubation of the left main coronary artery) ) Was then placed in the left main coronary artery under fluoroscopy, and after placing the catheter in place, it was placed in a first programmable syringe pump (eg, NE- 1000 Programmable Syringe Pump, New Era Pump Systems), then delivered AAV1 / SERCA2a, then dead volume of catheter and second programmable syringe pump And washed with sterile saline.

Results The results show a substantial improvement in one or more bioactivity / efficacy measures in subjects treated with AAV1 / SERCA2a and nitroglycerin compared to subjects treated with AAV1 / SERCA2a without nitroglycerin. SERCA2a expression is shown in heart section shown in Figure 1 at the end of sacrifice at day 30 (cardiac section diagram for SERCA2a expression, A (LV basal layer), # 1 septum, # 2 LV anterior wall, # 3 LV free wall, # 4 LV rear wall, B (LV middle layer), # 5 septum, # 6 LV front wall, # 7 LV free wall, # 8 LV rear wall, C (LV front layer), # 9 LV anterior wall, # 10 LV posterior wall, (RV), # 11 RV free wall basal layer, # 12 RV free wall tip layer) (LV free wall, LV posterior (lower) segment, and LV anterior) (Segment), RT-PCR (mRNA) and Western blot (protein). Calculate the overall mRNA and protein expression of individual sections in the left ventricle (sum of sections 1-10 in FIG. 1), septum (section # 5 in FIG. 1), and anterior middle wall (FIG. 1 Section # 6) was also analyzed separately. This is because these regions represent the deep myocardium farthest from the injection site. Obtaining appropriate gene transfer into these deep myocardial regions is most difficult, however, administration of nitroglycerin specifically increased vector uptake into these regions. The following results show improved SERCA2a mRNA and protein expression, particularly when AAV1 / SERCA2a is administered in combination with nitroglycerin in the LV anterior wall and septum. See Figures 2 and 3, respectively. In addition to normal background levels of SERCA2a mRNA and protein, increases in mRNA and protein are shown in these figures. This is in contrast to humans suffering from heart failure where SERCA2a expression levels are below normal levels. Therefore, the impact of AAV1 / SERCA2a treatment on SERCA2a expression levels in patients with heart failure is expected to be even more pronounced than the effects reported in Figures 2 and 3, considering the low baseline level of SERCA2a expression .

The mean aortic pressure from safety groups 3 and 4 is shown in FIG. The changes seen are most of the changes that were expected over the course of the experiment. There was a maximum 6 mmHg reduction in mean aortic pressure (MAP) compared to baseline, and all other changes for nitroglycerin treated animals were within the same range except for one hour in group 4 Please note that. This modest drop in MAP is not meaningful and does not cause safety problems. By comparison, Sasano et al. Reported a 30 mm Hg blood pressure drop after pretreatment and infusion of virus solution. In addition, subjects under Sasano's trial experienced a decrease in heart rate that averaged about 50-60 / min. The authors report that blood pressure and heart rate stabilized within 1 minute of perfusion. Nevertheless, ventricular fibrillation (VF) occurred at a rate of 50% during the intracoronary infusion for the first 10 pigs, but dropped to 5.6% for the remaining 71 pigs.

[Example 2]
Preliminary Phase I Clinical Trial Results-Effects of Nitroglycerin on Endpoint Measurement of Cardiac Function in Patients Given MYDICAR®

Results Table 2 below shows the tentative results of this study. These results represent a significant increase in the primary endpoint of one or more activities / efficacy in subjects treated with MYDICAR® and nitroglycerin compared to subjects treated without MYDICAR® nitroglycerin and Demonstrate significant improvement. For other details, see Hajjar et al., Journal of Cardiac Failure, 2008 14 (5); 355-67, the entire contents of which are incorporated herein by reference.

Claims (54)

  1.   A therapeutic polynucleotide for use in a method of treating or preventing cardiovascular disease by transfecting a large mammalian heart cell, said method prior to administration of said therapeutic polynucleotide, and / or Alternatively, a therapeutic polynucleotide comprising the step of dilating a blood vessel in the coronary circulation by administering a vasodilatory substance to the mammal simultaneously with the administration.
  2.   Administering the therapeutic polynucleotide to a coronary circulation vessel in vivo, wherein the therapeutic polynucleotide is infused into the vessel for at least about 3 minutes, wherein the coronary circulation is separated from the mammalian systemic circulation or 2. The therapeutic polynucleotide of claim 1, wherein the cardiovascular disease is treated or prevented by transfecting the therapeutic polynucleotide into the mammalian heart cells that are not substantially separated.
  3.   3. The therapeutic polynucleotide according to claim 2, wherein the vasodilator is a nitric oxide (NO) increasing substance.
  4.   Administering the NO-enhancing substance in a format selected from the group consisting of before the injection of the therapeutic polynucleotide, simultaneously with the injection of the therapeutic polynucleotide, and before and simultaneously with the injection of the therapeutic polynucleotide; The therapeutic polynucleotide according to claim 3.
  5.   The therapeutic polynucleotide according to claim 4, wherein the NO-enhancing substance is administered to a coronary blood vessel.
  6.   The NO enhancing agent is administered as a bolus injection within 5 minutes prior to the infusion of the therapeutic polynucleotide, and the NO enhancing agent is infused into the blood vessel simultaneously with the infusion of the therapeutic polynucleotide for at least about 10 minutes The therapeutic polynucleotide according to claim 5.
  7.   6. The therapeutic polynucleotide of claim 5, wherein the NO enhancer is about 50 μg to about 150 μg of nitroglycerin.
  8.   The administration of the NO-enhancing substance comprises an antegrade epicardial coronary injection of 1.5 mL of 100 μg / mL nitroglycerin solution into at least one of the left or right coronary artery via a percutaneous catheter for less than 1 minute The treatment of claim 3, wherein the administration of the NO-enhancing substance is less than 3 minutes prior to the infusion of the therapeutic polynucleotide and no other vasodilator or vascular penetration enhancer is administered to the mammal. Polynucleotide for use.
  9.   9. The therapeutic polynucleotide of claim 8, wherein the method further comprises injecting nitroglycerin into the blood vessel simultaneously with the infusion of the therapeutic polynucleotide.
  10. The mammal is a human, the cardiovascular disease is heart failure, the therapeutic polynucleotide is packaged in DNase resistant particles (DRP) of an AAV2 / 1 viral vector, and the total number of DRP injected into the blood vessel is about does not exceed 1 × 10 13, the therapeutic polynucleotide comprises a SERCA2a coding sequence, wherein the vessel is at least one of the left or right coronary artery, the injection of the therapeutic polynucleotide lasts for a period of at least about 10 minutes, claim The therapeutic polynucleotide according to 8.
  11.   The method of treatment or prevention improves the measure of absolute ejection fraction of the human heart 6 months after the treatment, as compared to the measure of absolute ejection fraction of the human heart before the treatment. The therapeutic polynucleotide according to claim 10.
  12.   4. The therapeutic polynucleotide according to claim 3, wherein the NO-enhancing substance is administered systemically in a format selected from the group consisting of intravenous injection, intravenous infusion, oral administration, transdermal administration, and subcutaneous administration.
  13.   The administration of the NO enhancer comprises administering about 0.5 mg to about 2.5 mg nitroglycerin by intravenous infusion over at least 30 minutes prior to the infusion of the therapeutic polynucleotide; 13. The therapeutic polynucleotide of claim 12, wherein the infusion begins within 3 minutes of completion of the intravenous infusion of nitroglycerin and no other vasodilator or vascular penetration enhancer is administered to the mammal.
  14.   14. The therapeutic polynucleotide of claim 13, wherein the method further comprises injecting an additional amount of nitroglycerin simultaneously with the infusion of the therapeutic polynucleotide.
  15. The mammal is a human, the cardiovascular disease is heart failure, the therapeutic polynucleotide is packaged in DNase resistant particles (DRP) of an AAV2 / 1 viral vector, and the total number of DRP injected into the blood vessel is about does not exceed 1 × 10 13, the therapeutic polynucleotide comprises a SERCA2a coding sequence, wherein the vessel is at least one of the left or right coronary artery, the injection of the therapeutic polynucleotide lasts for a period of at least about 10 minutes, claim 14. The therapeutic polynucleotide according to 13.
  16.   The method of treatment or prevention improves the measure of absolute ejection fraction of the human heart 6 months after the treatment, as compared to the measure of absolute ejection fraction of the human heart before the treatment. 16. The therapeutic polynucleotide of claim 15.
  17.   The therapeutic polynucleotide according to any one of claims 1 to 6 and 12, wherein the NO-enhancing substance is nitroglycerin.
  18.   Use of a therapeutic polynucleotide for the manufacture of a medicament for the treatment or prevention of cardiovascular disease in a large mammal, wherein the heart is obtained by transfecting said large mammalian heart cell with said therapeutic polynucleotide. A use wherein a vascular disease is treated or prevented and the medicament is for combined administration with a vasodilator that dilates the blood vessels in the coronary circulation of the mammal prior to and / or at the same time as administration of the medicament.
  19.   Administering the therapeutic agent to a coronary circulation blood vessel in vivo, wherein the therapeutic polynucleotide is infused into the blood vessel for at least about 3 minutes, wherein the coronary circulation is from the mammalian systemic circulation. 19. Use according to claim 18, wherein the use is separated or not substantially separated.
  20.   20. The use according to claim 19, wherein the vasodilatory substance is a nitric oxide (NO) increasing substance.
  21.   Administering the NO-enhancing substance in a format selected from the group consisting of before the injection of the therapeutic polynucleotide, simultaneously with the injection of the therapeutic polynucleotide, and before and simultaneously with the injection of the therapeutic polynucleotide; The use according to claim 20, wherein:
  22.   The use according to claim 21, wherein the NO-enhancing substance is administered to the blood vessels of the coronary circulation.
  23.   The NO enhancing agent is administered as a bolus injection within 5 minutes prior to the infusion of the therapeutic polynucleotide, and the NO enhancing agent is infused into the blood vessel simultaneously with the infusion of the therapeutic polynucleotide for at least about 10 minutes 23. Use according to claim 22.
  24.   23. Use according to claim 22, wherein the NO enhancing substance is about 50 [mu] g to about 150 [mu] g nitroglycerin.
  25.   The administration of the NO-enhancing substance includes an antegrade epicardial coronary injection of 1.5 mL of a 100 μg / mL nitroglycerin solution into at least one of the left or right coronary artery via a percutaneous catheter for less than 1 minute 21. Use according to claim 20, wherein the administration of the NO enhancing substance is less than 3 minutes prior to the infusion of the therapeutic polynucleotide and no other vasodilator or vascular penetration enhancer is administered to the mammal. .
  26.   26. The use according to claim 25, further comprising injecting nitroglycerin into the blood vessel simultaneously with the infusion of the therapeutic polynucleotide.
  27. The mammal is a human, the cardiovascular disease is heart failure, the therapeutic polynucleotide is packaged in DNase resistant particles (DRP) of an AAV2 / 1 viral vector, and the total number of DRP injected into the blood vessel is about does not exceed 1 × 10 13, the therapeutic polynucleotide comprises a SERCA2a coding sequence, wherein the vessel is at least one of the left or right coronary artery, the injection of the therapeutic polynucleotide lasts for a period of at least about 10 minutes, claim Use as described in 25.
  28.   The method of treatment or prevention improves the measure of absolute ejection fraction of the human heart 6 months after the treatment, as compared to the measure of absolute ejection fraction of the human heart before the treatment. 28. Use according to claim 27.
  29.   21. The use according to claim 20, wherein the NO enhancing substance is administered systemically in a form selected from the group consisting of intravenous injection, intravenous infusion, oral administration, transdermal administration, and subcutaneous administration.
  30.   The administration of the NO enhancer comprises administering about 0.5 mg to about 2.5 mg nitroglycerin by intravenous infusion over at least 30 minutes prior to the infusion of the therapeutic polynucleotide; 30. The use of claim 29, wherein the infusion begins within 3 minutes of completion of the intravenous infusion of nitroglycerin and no other vasodilator or vascular penetration enhancer is administered to the mammal.
  31.   32. The use of claim 30, further comprising injecting an additional amount of nitroglycerin simultaneously with the infusion of the therapeutic polynucleotide.
  32. The mammal is a human, the cardiovascular disease is heart failure, the therapeutic polynucleotide is packaged in DNase resistant particles (DRP) of an AAV2 / 1 viral vector, and the total number of DRP injected into the blood vessel is about does not exceed 1 × 10 13, the therapeutic polynucleotide comprises a SERCA2a coding sequence, wherein the vessel is at least one of the left or right coronary artery, the injection of the therapeutic polynucleotide lasts for a period of at least about 10 minutes, claim Use as described in 30.
  33.   The method of treatment or prevention improves the measure of absolute ejection fraction of the human heart 6 months after the treatment, as compared to the measure of absolute ejection fraction of the human heart before the treatment. Use according to claim 32.
  34.   30. Use according to any one of claims 18 to 23 and 29, wherein the NO enhancing substance is nitroglycerin.
  35. A method of treating or preventing cardiovascular disease by transfecting a large mammalian heart cell comprising:
    Identifying a mammal in need of treatment or prevention of cardiovascular disease;
    Administering to said mammal sufficient vasodilator to dilate coronary blood vessels; and
    administering a therapeutic polynucleotide to a coronary blood vessel in vivo,
    Injecting the therapeutic polynucleotide into the blood vessel for at least about 3 minutes, wherein the coronary circulation is not separated or substantially separated from the mammalian systemic circulation, and the therapeutic polynucleotide is injected into the mammalian heart cell; A method wherein the cardiovascular disease is treated or prevented by transfecting.
  36.   36. The method of claim 35, wherein the vasodilator is a nitric oxide (NO) augmenting substance.
  37.   38. The method of claim 36, wherein the NO enhancing substance is nitroglycerin.
  38.   40. The method of claim 36, wherein the NO enhancing agent is administered to a coronary blood vessel.
  39.   Administering the NO-enhancing substance in a format selected from the group consisting of before the injection of the therapeutic polynucleotide, simultaneously with the injection of the therapeutic polynucleotide, and before and simultaneously with the injection of the therapeutic polynucleotide; 40. The method of claim 38.
  40.   39. The method of claim 38, wherein the NO enhancing agent is administered as a bolus injection within 5 minutes prior to the infusion of the therapeutic polynucleotide.
  41.   The NO-enhancing substance is administered as a bolus injection within 5 minutes prior to the infusion of the therapeutic polynucleotide, and the NO-enhancing substance is injected into the blood vessel simultaneously with the infusion of the therapeutic polynucleotide for at least about 10 minutes 40. The method of claim 38.
  42.   40. The method of claim 38, wherein the NO enhancer is about 50 [mu] g to about 150 [mu] g nitroglycerin.
  43.   The administration of the NO-enhancing substance includes an antegrade epicardial coronary injection of 1.5 mL of a 100 μg / mL nitroglycerin solution into at least one of the left or right coronary artery via a percutaneous catheter for less than 1 minute 40. The method of claim 38, wherein the administration of the NO enhancing agent is less than 3 minutes prior to the infusion of the therapeutic polynucleotide and no other vasodilator or vascular penetration enhancer is administered to the mammal. .
  44.   44. The method of claim 43, further comprising injecting nitroglycerin into the blood vessel simultaneously with the infusion of the therapeutic polynucleotide.
  45. The mammal is a human, the cardiovascular disease is heart failure, the therapeutic polynucleotide is packaged in DNase resistant particles (DRP) of an AAV2 / 1 viral vector, and the total number of DRP injected into the blood vessel is about does not exceed 1 × 10 13, the therapeutic polynucleotide comprises a SERCA2a coding sequence, wherein the vessel is at least one of the left or right coronary artery, the injection of the therapeutic polynucleotide lasts for a period of at least about 10 minutes, claim 44. The method according to 43.
  46.   46. The treatment improves the absolute ejection fraction measurement of the human heart 6 months after the treatment as compared to the absolute ejection fraction measurement of the human heart prior to the treatment. The method described in 1.
  47.   38. The method of claim 36, wherein the NO enhancer is administered systemically.
  48.   48. The method of claim 47, wherein the NO enhancing agent is administered systemically in a form selected from the group consisting of intravenous injection, intravenous infusion, oral administration, transdermal administration, and subcutaneous administration.
  49.   Administering the NO-enhancing substance in a format selected from the group consisting of before the injection of the therapeutic polynucleotide, simultaneously with the injection of the therapeutic polynucleotide, and before and simultaneously with the injection of the therapeutic polynucleotide; 49. The method of claim 48, wherein:
  50.   48. The method of claim 47, wherein the NO enhancer is nitroglycerin.
  51.   About 0.5 mg to about 2.5 mg of nitroglycerin is administered by intravenous infusion for at least 30 minutes prior to the infusion of the therapeutic polynucleotide, the infusion of the therapeutic polynucleotide being an intravenous infusion of the nitroglycerin. 52. The method of claim 50, wherein no other vasodilator or vascular penetration enhancer is administered to the mammal beginning within 3 minutes of termination.
  52.   52. The method of claim 51, further comprising injecting an additional amount of nitroglycerin concurrently with the infusion of the therapeutic polynucleotide.
  53. The mammal is a human, the cardiovascular disease is heart failure, the therapeutic polynucleotide is packaged in DNase resistant particles (DRP) of an AAV2 / 1 viral vector, and the total number of DRP injected into the blood vessel is about does not exceed 1 × 10 13, the therapeutic polynucleotide comprises a SERCA2a coding sequence, wherein the vessel is at least one of the left or right coronary artery, the injection of the therapeutic polynucleotide lasts for a period of at least about 10 minutes, claim 51. The method according to 51.
  54.   54. The treatment improves the absolute ejection fraction measurement of the human heart 6 months after the treatment as compared to the absolute ejection fraction measurement of the human heart prior to the treatment. The method described in 1.
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